Progenitor cell specification in the developing mammalian pancreas is initiated by signals from definitive endoderm and surrounding mesenchyme. The subsequent cascade of pancreas-specific transcription factors expressed within these progenitor cells then permits the differentiation of the three cell lineages: endocrine, exocrine and duct cells. Importantly, studies using animal models have determined that the multipotent pancreatic progenitor cells (MPCs) in the developing pancreas are characterized by the expression of the transcription factors Pdx1, Ptf1A and Sox9. From these self-renewing MPCs all cells of the mature pancreas, including the insulin-producing ? cells, can be generated. Whereas most of our knowledge of the processes and factors involved in pancreatic organogenesis has been derived from animal studies, in vitro systems using human pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells) have greatly assisted our ability to translate these findings to the human setting. However despite these advances, the isolation of endogenous human MPCs remains elusive. The goal of our project is to purify and define the endogenous human MPC population. Our preliminary observations reveal the presence of cells expressing PDX1 and PTF1A that also co-express SOX9/FGFR2 within the human pancreas. These cells are located in the expanding branches of the developing epithelium. Most importantly, we have developed a method to isolate live MPCs expressing PTF1A, SOX9 and FGFR2 from human fetal pancreas using a fluorescent RNA probe technology in combination with fluorescence activated cell sorting. We hypothesize that the PTF1A+SOX9+ FGFR2+ cells represent a MPC population within the human pancreas that can give rise to all lineages, including endocrine, exocrine and ductal. We will use our innovative sorting technology to characterize by RNA-seq the endogenous human MPC population and confirm their differentiation capability. The possibility of establishing a genetic ?signature? for MPCs directly isolated from the human pancreas will have great impact, not only in the field of pancreas development, but also on the field of regenerative medicine where culture-based differentiation protocols are being developed to generate insulin- producing ? cells in vitro for therapeutic purposes. Ultimately, the development of technologies to isolate and culture human MPC represents an important tool for the identification of novel targets that can then be exploited to advance the development of translational approaches for the generation of cellular therapy for individuals with diabetes.